PROJECT SUMMARY Head and neck squamous cell carcinoma (HNSCC) is the seventh leading cause of cancer-related deaths worldwide, and thus far the genomic alterations identified in this disease have not had an impact on clinical care. Our group was among the first to report frequent inactivating mutations of NOTCH1 in HNSCC. More recently, the incidence of NOTCH1 mutations was found to be roughly 20% among over 500 patient HNSCC tumors sequenced as part of The Cancer Genome Atlas (TCGA) project, placing NOTCH1 among the top five most frequently mutated genes in this cancer type. HNSCC cell lines harboring NOTCH1 mutations are significantly more sensitive to six different drugs targeting the PI3K/mTOR pathway than HNSCC cell lines with wild-type (wt) NOTCH1. Unlike HNSCC tumors with PIK3CA mutations, which exhibited only growth arrest after treatment with PI3K/mTOR inhibitors, cell lines harboring NOTCH1 mutation also underwent cell death. Proteomic profiling of drug-sensitive HNSCC cell lines harboring NOTCH1 mutations and drug-resistant HNSCC lines with wt NOTCH1 before and after drug treatment revealed no differences in the modulation of many drug targets directly downstream of PI3K, including AKT. However, NOTCH1 mutants experienced greater drug-induced decreases in total expression of 3-phosphoinositide dependent kinase 1 (PDK1) and Aurora kinase B. Collectively, these data led to the hypothesis that HNSCC tumors with NOTCH1 mutations are highly sensitive to PI3K inhibitors because the PI3K signaling pathway is uniquely tied to regulation of total PDK1 protein levels, impacting both phosphatidylinositol-dependent and -independent PDK1 function in this genomic subtype. Therefore, drugs targeting PI3K/mTOR should be effective for treating HNSCC patients with NOTCH1-mutant tumors. We will address these hypotheses with a clinical trial (Aim 1) and conduct in vitro mechanistic studies (Aim 2) and preclinical studies to identify ways to enhance killing of HNSCC tumors harboring NOTCH1 mutations through combination therapy (Aim 3). The proposed research will have a positive impact because it will be the first to establish a therapeutic vulnerability of NOTCH1-mutant HNSCC to any class of drugs and may inform the development of the first biomarker-driven targeted therapy for HNSCC. Additionally, we may define a previously unknown mechanism of PDK1 regulation and identify clinically relevant targets that enhance the efficacy and durability of PI3K inhibition in NOTCH1-mutant HNSCC. Because NOTCH1 loss-of-function mutations are common in other squamous cell carcinomas, including those of the skin, esophagus, and lung, these findings will likely have implications beyond HNSCC.